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Mitochondrial manganese superoxide dismutase (MnSOD), encoded by the SOD2 gene, represents a major cellular defense against environmental carcinogens that cause oxidative stress. Two single-nucleotide polymorphisms −9 T>C (V16A in the MnSOD mitochondrial targeting sequence) and −102 C>T (in the SOD2 promoter sequence) modify risk toward various types of malignancies and overall survival. Since little is known about the effects of these polymorphisms on overall enzyme function in normal human tissue, the goal of this study was to evaluate their functional effects in cryopreserved human hepatocytes. Cryopreserved human hepatocytes were genotyped for the MnSOD −9 T>C and −102 C>T polymorphisms by TaqMan allelic discrimination assays. MnSOD catalytic activities were determined in vitro in lysates derived from the hepatocytes. In random samplings of cryopreserved hepatocytes, 16% possessed the −9 T>C and 6% possessed polymorphism on at least one of the two alleles. −9 T>C (V16A) significantly (p<0.02) reduced MnSOD catalytic activity whereas −102 C>T did not (p>0.05). The −9 T>C (V16A) polymorphism in the MnSOD mitochondrial targeting sequence significantly reduced MnSOD catalytic activity in cryopreserved hepatocytes, consistent with its reported associations with cancer risk and treatment.
Mitochondrial reactive oxygen species (ROS) are detoxified by the successive action of manganese superoxide dismutase (MnSOD) and glutathione peroxidase. MnSOD dismutates the superoxide anion into hydrogen peroxide (H2O2), which glutathione peroxidase detoxifies in to water (Wallace, 1999).
There are three distinct types of superoxide dismutase (SOD) activity found in human cells: (1) a homodimeric cytosolic Cu/Zn-SOD (McCord and Fridovich, 1969), (2) an extracellular homotetrameric glycosylated SOD (Marklund, 1982), and (3) a mitochondrial matrix homotetrameric MnSOD (Weisiger and Fridovic, 1973). Numerous reports indicate a relative deficiency of mitochondrial MnSOD in human tumors (Oberley and Oberley, 1984). Interest in this relative deficiency of SOD activity has been greatly increased by observations that overexpression of SOD in tumor cells will suppress cell division in culture and tumor growth in vivo (St. Clair et al., 1997). While the precise reasons for this relationship between tumor cell growth rate and intracellular SOD activity are not known, these findings support the general idea that decreased expression of SOD may promote tumor growth. In fact, as a result of these observations, Oberley and Oberley (1984) have concluded that MnSOD acts as a tumor suppressor gene.
Further evaluation of MnSOD suggests that it is critically important in the maintenance of mitochondrial function. Mice with total deficiency of this enzyme were not viable, and even the heterozygotes exhibited progressive cardiomyopathy (Li et al., 1995). These studies further showed that transgenic mice that express human MnSOD in the mitochondria are protected from environmental oxygen–induced lung injury (Wispe et al., 1992). In contrast, disruption of the other two SODs yielded viable mice, which were normal in nonstressful conditions (Yen et al., 1999). Thus, the mitochondrial MnSOD represents a major cellular defense against environmental carcinogens that cause oxidative stress.
MnSOD is encoded by nuclear DNA and is inducible by ROS, cytokines, cigarette smoke, and ethanol (Koch et al., 1994; Perera et al., 1995; Xu et al., 1999a). MnSOD is synthesized with a mitochondrial targeting sequence, which drives its mitochondrial import (Wispe et al., 1989; Sutton et al., 2003). In the matrix, the mitochondrial targeting sequence is cleaved, and the mature protein assembles into the active tetramer (Wispe et al., 1989; Sutton et al., 2003). A −9 T>C (V16A) genetic polymorphism in the mitochondrial targeting sequence results in substitution of alanine for valine. The Ala-MnSOD variant, whose presequence has an α-helix structure, is easily imported and achieves high mitochondrial activity, whereas the Val-MnSOD variant, whose presequence may have a partial β-sheet structure, is partly retained within the narrow inner membrane import pore (Sutton et al., 2003), and is partly degraded in the proteasome (Sutton et al., 2005). Further, its mRNA is rapidly degraded, so that the Val-MnSOD variant exhibits lower activity (Sutton et al., 2005).
The human MnSOD gene is characterized by a lack of TATA or CAAT boxes along with the presence of a GC-rich region containing multiple SP-1 and activator protein-2 (AP-2) binding sites (Wan et al., 1994). Further work identified one possible cause for the reduced expression of MnSOD in some (but not all) human tumor lines, the occurrence of three heterozygous mutations in the upstream promoter region of the MnSOD gene (Xu et al., 1999b). One of these mutations in the MnSOD promoter sequence (MnSOD −102 C>T) has been shown to change the binding pattern of AP-2, leading to a reduction in transcriptional activity (Xu et al., 1999b). However, the functional effect of this polymorphism in the MnSOD promoter sequence on enzymatic activity has not been reported. Thus the aim of this study was to evaluate the functional effects of the −9 T>C and −102 C>T MnSOD gene polymorphisms on catalytic activity in lysates derived from cryopreserved human hepatocytes.
Cryopreserved human hepatocytes were obtained from Celsus In Vitro Technologies (Baltimore, MD). They were thawed according to the manufacturer's instructions by warming a vial of the hepatocytes at 37°C for 90s, and transferring the contents to a 50mL conical tube containing 45mL of InVitroGRO HT medium. The cell suspension was centrifuged 50g at room temperature for 5min. The supernatant was discarded, and the cells were resuspended in 20mM NaPO4, 1mM dithiothreitol, 1mM EDTA, 1mM phenylmethylsulfonyl fluoride, 1μM pepstatin A, and 1μg/mL aprotin and stored at −70°C. Hepatocytes were lysed by exposing the cells to three freeze/thaw cycles at −70°C and 37°C. The lysate was spun at 15,000g for 20min, and the supernatant was aliquoted and stored at −70°C.
DNA was isolated using the QIAamp DNA mini kit from Qiagen (Valencia, CA) following manufacturer's instructions. In brief, appropriate number of cells (maximum 5×106 cells) were centrifuged for 5min at 300g in a 1.5mL microcentrifuge tube to remove the supernatant completely. The cell pellet was resuspended in PBS, and proteinase K was added to lyse the cells. DNA was purified using a QIAamp spin column and eluted with water.
Protein concentrations in the lysates were determined with the Bio-Rad protein assay kit (Bio-Rad, Richmond, CA).
The −9 T>C and −102 C>T MnSOD polymorphisms were determined by a TaqMan allelic discrimination assay as recently published (Martin et al., 2004, 2005). SNP-specific polymerase chain reaction (PCR) primers and fluorogenic probes were designed using Primer Express (Version 1.5; Applied Biosystems, Foster City, CA). The fluorogenic probes are labeled with a reporter dye (either carboxyfluorescein [FAM] or VIC) and are specific for one of the two possible bases (−9 T or C) and (−102 C or T) in the MnSOD gene. TaqMan Universal PCR Master Mix (Applied Biosystems) was used to prepare the PCR. The 2× mix was optimized for TaqMan reactions and contained AmpliTaq-Gold DNA polymerase, AmpErase, uracil DNA glycosylase, dNTPs with UTP, and a passive reference. Primers, probes, and genomic DNA were added to final concentrations of 300μM, 100μM, and 0.5–2.5ng/μL, respectively. Controls (no DNA template) were run to ensure that there was no amplification of contaminating DNA. The amplification reactions were carried out in an ABI Prism 7700 Sequence Detection System (Applied Biosystems) with two initial hold steps (50°C for 2min, followed by 95°C for 10min) and 50 cycles of a two-step PCR (95°C for 15s and 60°C for 1min). The fluorescence intensity of each sample was measured at each temperature change to monitor amplification of the region of the MnSOD gene of interest. The nucleotide present at the SNP position is determined by the fluorescence ratio of the two SNP-specific fluorogenic probes. The fluorescence signal increases when the probe with the exact sequence match binds to the single-stranded template DNA and is digested by the 5′-3′ exonuclease activity of AmpliTaq-Gold DNA polymerase (Applied Biosystems). Digestion of the probe releases the fluorescent reporter dye (either FAM or VIC) from the quencher dye.
MnSOD catalytic activity was determined with the SOD Assay Kit-WST (Dojindo Molecular Technologies, Gaithersburg, MD) according to the manufacturer's protocol. In brief, SOD catalytic activity was measured by utilizing a highly water-soluble tetrazolium salt, WST-1 (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfo-phenyl)-2H-tetrazolium, monosodium salt), which produces a water-soluble formazan dye upon reduction with a superoxide anion. MnSOD catalytic activity was determined in the presence of 1mM KCN to block Cu/Zn-SOD activity and normalized to total lysate protein.
Chi-square analysis was used to compare genotype and MnSOD catalytic activity. Differences of p<0.05 were considered significant. Statistical analysis was performed using JMP software (JMP; SAS Institute, Cary, NC).
In this set of 49 cryopreserved hepatocytes, representing a random population of liver organ donors, 16% possessed the −9 T>C and 6% possessed the −102 C>T polymorphism on at least one of the two alleles. Eighty-four percent (n=42) of the samples had wild-type genotype (TT) in the −9, and 92% (n=39) had the wild-type genotype (CC) in the −102. The −9 T>C (V16A) polymorphism in the mitochondrial targeting sequence significantly (p<0.02) reduced MnSOD catalytic activity in lysates of the cryopreserved hepatocytes, whereas no significant effect (p>0.05) was observed with the −102 C>T polymorphism (Fig. 1).
One of the first reports of MnSOD enzyme function in normal healthy individuals was demonstrated by Kawaguchi et al. (1990). This report utilized enzyme-linked immunosorbent assay to measure MnSOD function in 401 individuals whose liver function tests were normal. They demonstrated higher levels of MnSOD in patients with acute myocardial infarction as well as malignant diseases, such as acute myeloid leukemia, primary hepatoma, and gastric cancer. The authors did not evaluate the effects that various MnSOD polymorphisms have on these MnSOD activity levels.
A more recent report from Bastaki et al. (2006) evaluated erythrocyte MnSOD activities in 231 healthy nonsmoking student volunteers and compared them to individuals possessing the MnSOD −9 T>C (V16A) polymorphism in the mitochondrial targeting sequence. The authors utilized a relatively similar method (MnSOD activity kit; Kamiya Biomedical, Seattle, WA), and normalized MnSOD activities to hemoglobin content. Their study demonstrated a greater MnSOD activity in people possessing the homozygous (−9 T/T; 16V) and heterozygous (−9 T/C; 16V/A) genotypes compared to the homozygous variant genotype (−9 C/C; 16A). Thus, these results are consistent with our data showing the −9 T>C (V16A) polymorphism was associated with a significant decrease of MnSOD activity in cryopreserved human hepatocytes.
However, conflicting data have been reported from Sutton et al. (2003), who based on computer modeling predicted a partial α-helix structure for the Ala substitution, but a β-sheet structure for the Val substitution, which could hamper mitochondrial import. They evaluated an in vitro import model (Sutton et al., 2003) and HuH7 human hepatoma cell line (Sutton et al., 2005) confirming their hypothesis that the Val substitution leads to decreased contranslational import and thus decreased activity. Although we do not understand the reasons for this discrepancy, we believe that our results in human hepatocytes and the results of Bastaki et al. in human erythrocytes (2006) represent more clinically relevant models.
Our results and others do imply that the effects of the −9 T>C polymorphism is related to a transport efficiency into the mitochondria. As presented by DeGoul et al. (2001), there is a significant greater risk of severe alcoholic cirrhosis in patients with the −9 T>C polymorphism when compared to wild type when alcohol consumption is the same for both groups. The mitochondria are the main source of ROS in the normal healthy cell, and the burden of ROS is increased with ethanol consumption (Kukielka et al., 1994), smoking, and other environmental factors. The mitochondrial respiratory chain initially forms the superoxide anion radical, which the MnSOD reduces to H2O2, which is detoxified into water by mitochondrial glutathione peroxidase (Shigenaga et al., 1994). This delicate balance of both detoxifying enzymes is required, and the effects of too little and too much MnSOD activity can have a detrimental effect leading to either increased superoxide ion radical if too little or increased hydroxyl radical if too much MnSOD activity (Li et al., 1998; Neuman et al., 1998; Esposito et al., 2000). These dual effects of MnSOD maybe the reason for conflicting reports from Sutton and our data, but more transport efficiency studies are needed. Similarly, there are probable other polymorphisms in the transport coding sequence and other polymorphisms in other detoxifying genes that also play a role in overall effect of MnSOD activity. Lastly, the transport efficiency and activity of MnSOD may differ in the noncancerous normal tissue, normal tissue in a cancer specimen, and cancer tissue itself. All three are different in the MnSOD activity, and further evaluations of these concepts are needed.
As with the results from the enzymatic activity, there are also mixed results reported for the effects of both MnSOD −9 T>C and −102 C>T polymorphisms on disease risk and treatment. In evaluations of the MnSOD −9 T>C genotype, there are reports demonstrating an increase in breast cancer incidence or decrease in overall survival with the Ala substitution (Ambrosone et al., 1999; Mitrunen et al., 2001; Cebrian et al., 2006; Cox et al., 2006). However, higher risk of lung cancer has been reported for both the Val (Wang et al., 2001) and the Ala (Liu et al., 2004a, 2004b) substitutions.
In an evaluation of the −102 C>T genotype, less has been published, since its presence was only recently added to the GenBank reference list Genebank Bankit #574003, AY397775. In an evaluation of gastric cancer, the association of the MnSOD polymorphisms at −102 C>T was not found to be associated with gastric cancer in a Polish case–control study (Martin et al., 2005). However in an evaluation of breast cancer, the MnSOD −102 C>T variant allele appears to be associated with an improved recurrence-free survival in all patients, and more dramatically in subjects who received adjuvant radiation therapy (Martin et al., 2006).
Thus, in summary, our results provide insight into the role of genetic polymorphisms affecting MnSOD expression as potential markers for disease diagnosis or treatment.
This study was supported by the grant from National Cancer Institute R01-CA04627.
No competing financial interests exist.